Design, synthesis and anticancer evaluation of polymethoxy aurones as potential cell cycle inhibitors

Abstract

Background

Cancer is the most fatal disease in humans and the aberrant activity of various cell cycle proteins results in uncontrolled tumor cell proliferation, thus, regulating the cell cycle is an attractive target in cancer therapy.

Objectives

Aurone is a naturally occurring active compound with a wide range of biological activities, of which 3, 4, 5-trimethoxyphenyl (TMP) is an important microtubule targeting pharmacophore. Based on the pharmacophore combination principle, we incorporate the TMP pharmacophore into the aurone structure and design a novel polymethoxy derivative that is expected to inhibit tumor cell proliferation through regulating the cell cycle.

Methods

By introducing different substituents on C-4′ and C-3′, a series of new 4, 5, 6-trimethoxy aurone derivatives have been designed and synthesized. DU145, MCF-7 and H1299 cell lines were selected to evaluate their anticancer activity. The compound with the best cytotoxicity was then selected and the anticancer mechanisms were investigated by network pharmacology, flow cytometry, Western blot, and cell heat transfer assay. ADMET prediction evaluated the draggability of aurone derivatives.

Results

Aurones 1b and 1c have selective anti-proliferative activity against DU145 cells. Among them, the compound 1c have better cytotoxicity against DU145. Compound 1c could bind the active cavity of CyclinB1/CDK1/CKS complex protein and induced G2/M phase arrest of DU145 cells by regulating the expression of CyclinB1 and p21. Compound 1c satisfies the Lipinski rule, is suitable for the absorption and metabolism index, and has a lower risk of cardiac toxicity.

Conclusions

Polymethoxy aurones 1c might function as a CyclinB1/CDK1 inhibitor that deserved to be further developed for the treatment of prostate cancer.

1. Introduction

Nowadays, cancer is the most fatal disease in humans, and the incidence and mortality are sharply growing in each year. Aberrant activity of various cell cycle proteins results in uncontrolled tumor cells proliferation, therefore, regulating the cell cycle is an attractive target in cancer therapy [1].

Aurones (2-benzylidenebenzofuran-3(2H)-ones), which are structurally isomeric of flavonoids, is the main ingredient conferring to some bright golden flowers [2]. Aurones have been reported to show abroad bioactivities, such as antitumor [3], antioxidative [4], and antiviral [5] activities. Especially, aurone is a novel skeleton as an anticancer agent inhibiting the DNA strand-scission, topoisomerase, cyclin-dependent kinase (CDKs) and tubulin polymerization activities [6]. Moreover, the 3, 4, 5-trimethoxyphenyl (TMP) is also an important moiety in microtubule-targeting agents (MTAs), such as combretastatin A-4 (CA-4) have achieved significant success in the treatment of cancer. [7,8] Following the pharmacophore combination principle, we introduced three trimethoxy groups into the A-ring of the aurone and added different groups in the B-ring to design a series of novel anti-tumor molecules that are expected to inhibit tumor cell proliferation through the cell cycle (Fig. 1).

Fig. 1.

The design of 4, 5, 6-trimethoxy aurone derivatives through the pharmacophore combination principle.

2. Materials and methods

2.1. General information

Melting points were determined using X-5 digital display binocular microscope (uncorrected). ¹H NMR and ¹³C NMR spectra were recorded on a Bruker Avance Ⅲ HD600 (600 MHz) spectrometer; J values are in Hz. Mass spectra (Water Xevo G2-XS qTOF) were recorded on an electro-spray ionization mass spectrometer as the value m/z.

Western blot on the membrane were developed with the ECL system (Thermo Fisher, USA) and images were captured using the Odyssey infrared imaging system (LI-COR Biosciences, USA). Band intensity was quantified by densitometric analysis using Image Studio Lite software (LI-COR Biosciences, USA). Cell cycle distribution was evaluated using a flow cytometer (BD LSR Fortessa, Becton Dickinson & Co., USA).

Human prostate cancer (DU145), breast cancer (MCF-7) and lung cancer (H1299) cell lines were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China). Phosphate buffered saline (PBS), Roswell Park Memorial Institute (RPMI)-1640 medium, Dulbecco's modifcation of Eagle's medium (DMEM) medium, penicillin/streptomycin, and antibiotics were purchased from Beijing Vicente Biotechnology Co., Ltd. (Beijing, China). Dimethyl sulfoxid (DMSO) was obtained from Sigma-Aldrich (St.Louis, MO, USA). Fetal calf serum (FBS) was obtained from Gibco (Grand Island, NY, USA). Cell Counting Kit-8 (CCK-8) Assay Kit was obtained from Boster Biological Technology co. Itd (Wuhan, China). Cell Cycle Kit was purchased from BD Biosciences (San Jose, CA, USA). Radioimmunoprecipitation assay (RIPA) buffer were purchased from Thermo Fisher Scientifc (Waltham, USA). BCA (Bicinchoninic Acid) Protein Assay Kit was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). PVDF (polyvinylidene fluoride) membranes purchase from Millipore, USA. All the primary antibodies such as p21, CDK1, CyclinB1 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were purchased from Cell Signaling Technology (Danvers, USA).

2.2. Chemistry

2.2.1. General procedure for the synthesis of aurones (compound 1)

To a solution of 2 (51.2 mg, 0.23 mmol) in methanol (10 mL) was added 80 % KOH (0.08 mL). The solution was stirred at room temperature for 0.5 h. Then, substituted benzaldehyde (0.76 mmol) was added dropwise, and the mixture refluxed for 1–30 h (monitored by TLC). Cooled to room temperature, remove methanol under reduced pressure. Added water to the residue, and acidized by HCl, then precipitate appears. Filtered the solid under vacuum to give the crude product. The crude product was purified by recrystallization or silica column chromatography [9].

2.2.2. (Z)-2-(4-hydroxybenzylidene)-4,5,6-trimethoxybenzofuran-3(2H)-one (compound 1a)

Silica column chromatography (EtOAc/petroleum ether, v/v, 1:4) to give yellow solids, yield 68.2 mg (90.4 %), mp 204–205 °C. ¹H NMR (600 MHz, CDCl₃) δ, ppm (J, Hz): 3.85(3H, s, –OCH₃), 4.00(3H, s, –OCH₃), 4.28(3H, s, –OCH₃), 6.57(1H, s, Ph-H), 6.77(1H, s, –CH), 6.93–6.94(2H, d, J = 6.0 Hz, Ph-H), 7.81–7.82(2H, d, J = 6.0 Hz, Ph-H). ¹³C NMR (150 MHz, CDCl₃) δ, ppm: 180.98, 163.85, 161.56, 157.55, 151.61, 146.51, 136.59, 133.28, 125.03, 116.03, 111.93, 107.34, 90.55, 62.38, 61.66, 56.63. Found, m/z: 329.1020 [M+H]⁺. C₁₈H₁₆O₆, Calculated, m/z: 329.1028.

2.2.3. (Z)-2-(3-chloro-4-hydroxybenzylidene)-4,5,6-trimethoxybenzofuran-3(2H)-one (compound 1b)

Silica column chromatography (EtOAc/petroleum ether, v/v, 1:4) to give yellow solids, yield 35.8 mg (43.0 %), mp 193–194 °C. ¹H NMR (600 MHz, CDCl₃) δ, ppm (J, Hz): 3.85(3H, s, –OCH₃), 4.02(3H, s, –OCH₃), 4.28(3H, s, –OCH₃), 6.60(1H, s, Ph-H), 6.68(1H, s, –CH), 7.10–7.11(1H, d, J = 12.0, Ph-H), 7.67–7.69(1H, d, J = 12.0, Ph-H), 7.95(1H, s, Ph-H). ¹³C NMR (150 MHz, CDCl₃) δ, ppm: 180.65, 163.88, 161.70, 152.42, 151.73, 147.08, 136.72, 131.81, 131.47, 126.35, 120.55, 116.65, 109.83, 107.07, 90.62, 62.40, 61.65, 56.69. Found, m/z: 363.0630 [M+H]⁺. C₁₈H₁₅ClO₆, Calculated, m/z: 363.0634.

2.2.4. (Z)-2-(3-bromo-4-hydroxybenzylidene)-4,5,6-trimethoxybenzofuran-3(2H)-one (compound 1c)

Silica column chromatography (EtOAc/petroleum ether, v/v, 2:7) to give yellow solids, yield 39.2 mg (42.0 %), m. p. 207–208 °C. ¹H NMR (600 MHz, CDCl₃) δ, ppm (J, Hz): 3.82(3H, s, –OCH₃), 4.00(3H, s, –OCH₃), 4.27(3H, s, –OCH₃), 6.57(1H, s, Ph-H), 6.65(1H, s, –CH), 7.07–7.09(1H, d, J = 12.0, Ph-H), 7.70–7.71(1H, d, J = 6.0, Ph-H), 8.01(1H, s, Ph-H). ¹³C NMR (150 MHz, CDCl₃) δ, ppm: 180.61, 163.88, 161.68, 153.25, 151.73, 147.08, 136.73, 134.49, 132.48, 126.82, 116.43, 110.84, 109.60, 107.07, 90.63, 62.41, 61.65, 56.70. Found, m/z: 407.0125 [M+H]⁺. C₁₈H₁₅BrO₆, Calculated, m/z: 407.0134.

2.2.5. (Z)-2-(4-bromobenzylidene)-4,5,6-trimethoxybenzofuran-3(2H)-one (compound 1d)

Recrystallized from ethanol to afford compound 1d, yellow solids, yield 43.2 mg (48.0 %), mp 187–189 °C. ¹H NMR (600 MHz, CDCl₃) δ, ppm (J, Hz): 3.82(3H, s, –OCH₃), 3.99(3H, s, –OCH₃), 4.25(3H, s, –OCH₃), 6.55(1H, s, Ph-H), 6.69(1H, s, –CH), 7.55–7.56(2H, d, J = 6.0 Hz, Ph-H), 7.71–7.73(2H, d, J = 12.0 Hz, Ph-H). ¹³C NMR (150 MHz, CDCl₃) δ, ppm: 180.77, 163.82, 161.41, 160.69, 151.65, 146.61, 136.60, 132.96, 125.28, 114.40, 111.49, 107.31, 90.52, 62.42, 61.64, 56.61. Found, m/z: 391.0176 [M+H]⁺. C₁₈H₁₅BrO₅, Calculated, m/z: 391.0183.

2.2.6. (Z)-2-(4-fluorobenzylidene)-4,5,6-trimethoxybenzofuran-3(2H)-one (compound 1e)

Recrystallized from ethanol to afford compound 1e, yellow solids, yield 20.0 mg (25.0 %), mp 202–203 °C. ¹H NMR (600 MHz, CDCl₃) δ, ppm (J, Hz): 3.85(3H, s, –OCH₃), 4.01(3H, s, –OCH₃), 4.28(3H, s, –OCH₃), 6.57(1H, s, Ph-H), 6.76(1H, s, –CH), 7.14–7.17(2H, t, J = 6.0 Hz, Ph-H), 7.88–7.90(2H, m, Ph-H). ¹³C NMR (150 MHz, CDCl₃) δ, ppm: 180.75, 164.02, 164.00, 162.33, 161.73, 151.78, 147.35, 147.33, 136.73, 133.11, 133.05, 128.83, 128.81, 116.09, 115.95, 110.00, 107.01, 90.54, 62.41, 61.64, 56.67. Found, m/z: 331.0976 [M+H]⁺. C₁₈H₁₅FO₅, Calculated, m/z: 331.0982.

2.3. Biological experiments

Microtubule-targeting inhibitors such as paclitaxel are broad-spectrum anti-tumor drugs which commonly used to treat solid tumors such as breast cancer, prostate cancer and lung cancer etc. For the past few years, we have focused our research efforts on screening antitumor drugs from natural products [10,11].

DU145 is an androgen independent cell with low differentiation and moderate metastatic potential and is considered as a standard prostate cancer cell line for therapeutic research [12]. Our previous studies showed that Marsdenia tenacissima (Roxb.) moon injection induced apoptosis of DU145 cell by regulating AKT/GSK3β/STAT3 signaling axis and exerted a potential anti-tumor effect in prostate cancer through inhibiting ErbB2-GSK3β-HIF1α signaling axis, which may be related to the tricarboxylic acid cycle [13,14]. Total flavonoids of Litchi seed attenuated the progression of prostate cancer including DU145 cells via inhibiting AKT/mTOR and NF-κB signaling pathways [15,16].

Breast cancer is the leading invasive cancer in women globally, being estrogen receptor-positive (ER+) the most common subtype [17]. MCF-7 is an estrogen receptor positive cell and retains multiple differentiated mammary epithelial characteristics [18,19]. Our previous studies revealed that chlorogenic acid inhibited epithelial-mesenchymal transition and invasion of MCF-7 cell by down-regulating LRP6 [20]. Total flavonoids of Litchi seed attenuated stem cell-like properties of breast cancer through reducing the percentage of CD44⁺CD24-/low cells, inhibiting the mammospheres formation and down-regulating the mRNA and protein levels of cancer stem cells related markers in MCF-7 cells by regulating Notch3 signaling pathway [21].

H1299 is an Non-small cell lung cancer cell line with p53-null [22] and is widely used in a variety of basic cell biology and biomedical studies, involving lung cancer proliferation, cell apoptosis, cell cycle [23], metastasis [24], and the development of inhibitors [25, 26, 27].

Therefore, in this paper, prostate cancer, breast cancer and lung cancer cell lines (DU145, MCF-7 and H1299) were selected for biological experiments.

2.3.1. Cell proliferation assay

DU145, MCF-7 and H1299 cell lines were cultured in RPMI-1640 media or DMEM media supplemented with 10 % FBS, 1 % penicillin/streptomycin and incubated in a 5 % CO₂ humidified atmosphere at 37 °C. CCK-8 assay was performed to assess cell proliferation ability followed the previous study [14]. DMSO was used as the mother liquor, and different concentrations of the solution were diluted with a complete medium. The absorbance values were measured at 450 nm. The Dose-response curves generation and IC₅₀ values calculation were performed by GraphPad Prism 7 [28]. The cytotoxic effects of the synthesized compounds on DU145, MCF-7 and H1299 cells were evaluated by CCK-8 method. The general procedure is as follows: cells are seeded in 96-well plates (2000–2500 cells/well) overnight, and then incubated with the compounds (1a-1e) at different concentrations (0.1–320 μM). After treatment for 72 h, the CCK-8 reagent was added to the culture medium and incubated with the cells for an additional 0.5 h. Absorbance was measured by a microplate reader (Thermo, USA) at 450 nm (OD1 means the optical density value in medicine groups, OD2 means the average optical density value in all duplicates in the control group). The relative cell viability in percentage was calculated as follows:

$$\text{Relative}\text{cell}\text{viability}(\%)=\frac{\text{OD}1}{\text{OD}2}\times 100\%$$

2.3.2. Network pharmacology

Disease-related target genes were obtained from GeneCards Database (https://www.genecards.org/)[29, 30], and the targets of compound were predicted by ChemMapper Database (http://lilab-ecust.cn/chemmapper/) [31]. Cytoscape software (version 3.2.1) was applied to graphically display the network of compound, disease and the targets interacting with them [32]. FunRich (version 3.1.3) was used for drawing a Venn diagram of targets both compound and disease related. To further explore the biological processes of the candidate compound involved, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (http://www.kegg.jp/) was applied to screen the top signal pathways based on the P-value.

2.3.3. Cell cycle assay

The DU145 cells were seeded into a cell culture dish (6 cm in diameter) at a density of 2.5 × 10⁵ cells/mL and incubated for 24 h at 37 °C and 5 % CO₂. Then, the medium was removed and replaced with a medium (DMSO 0.5 % v/v) containing 5 or 10 μM of compound 1c. After treatment for 72 h, the cell layer was trypsinized and washed with ice-cold phosphate buffered saline (PBS), fixed with 75 % ice-cold ethanol for overnight. The fixed cells were rinsed with PBS and then stained with the DNA fluorochrome 7-AAD in a solution containing stain buffer under dark condition for 15 min at 37 °C according to the instruction manual. Then samples were detected by flow cytometry (Accuri C6 Plus, BD Biosciences, United States).

2.3.4. Western blot analysis

The DU145 cells treated with compound 1c were lysed on ice using RIPA buffer to obtain total protein. Lysates were centrifuged at 12000 rpm for 15 min at 4 °C. The protein concentration was determined by a BCA protein assay kit. 40 μg of denatured protein samples was separated by SDS-PAGE (10 %–15 %) and transferred onto PVDF membranes. The PVDF membranes were further blocked with 5 % non-fat milk at room temperature for 1 h and then incubated with specific primary antibodies [p21, CDK1, CyclinB1 (working concentration 1:1000)] diluted in 1 × PBS at 4 °C overnight. After three washes with Tris-buffered saline Tween (TBST) for 15 min, the membranes were incubated with secondary antibodies (Anti-mouse IgG,1:30000). The results were presented as a percentage of the internal control GAPDH.

2.3.5. Molecular docking

Surflex-dock of SYBYL-X 2.0 software (Tripos Inc., St Louis, USA) was used to study the interaction between CyclinB1/CDK1/CKS and compound 1c. A ternary complex of human proteins CDK1/Cyclin B/CKS2 (PDB code:5HQ0 [33]) was selected and downloaded from the RCSB Protein Data Bank (https://www.rcsb.org/) [34], that the resolution is 2.30 Å.

Several protein preparation works were done before using the docking programs. Firstly, the molecular structure of compound 1c was optimized through the standard Tripos force field with a 0.005 kcal (mol Å)⁻¹ energy gradient convergence criterion and using Gasteiger–Hückel charges to calculate the partial atomic charges. The structural energy minimizations were performed using the Powell method 10000 iterations. The other parameters were set as default values in SYBYL-X 2.0 software. Secondly, the original ligand was extracted from the active site of the protein and all water molecules were removed from the original protein complex. Next, protein analysis and repair were carried out following the procedure demands, such as repair sidechain, termini treatment by charges state, set acids residues protonation type near ligands, added hydrogens, added charge by AMBER7 FF99 biopolymer and Gasteiger-Hückel ligands, fixed sidechain amides and staged minimization (minimization parameters were the same as molecular structure minimization). Afterward, the ProtoMol file was generated by the ligand-based docking mode and each structure was docked into the active pocket using surflex-dock. In addition, the co-crystallized ligand was set as a reference molecule during the docking simulation. Based on the similarity of the orientation between the original ligand and the conformations, the conformation with the highest docking score was chosen for the study of the binding mode.

2.3.6. Cellular thermal shift assay (CETSA)

DU145 cells were lysed by RIPA containing protease inhibitors and total proteins were collected. Cell lysates were centrifuged at 12,000 rpm for 20 min, the supernatant was equally divided into two parts, with one part treated with DMSO and the other part treated with 1c (200 μM). After incubation at room temperature for 1 h, the two parts were divided into seven aliquots (50 μL) respectively, then heated at temperatures of 37, 42, 47, 52, 57, 62, and 67 °C for 3 min. Afterward, every aliquot was centrifuged and the supernate was analyzed by Western blot.

2.3.7. Pharmacokinetics and drug likeness prediction

A successful drug should satisfy in biochemical properties, pharmacokinetics and safety. High potency and selectivity, desirable absorption, distribution, metabolism, excretion and toxicity (ADMET) characteristics were the important conditions for the success of a drug candidate [35, 36, 37]. Therefore, it is necessary to acknowledge the ADMET properties of the designed compounds in advance. We use the ADMET Evaluation function of ADMETlab 2.0 (https://admetmesh.scbdd.com/) to predict their ADMET properties [38].

The chemical structure of the compound is plotted in the web tool and then submitted. The analysis of absorption, distribution, metabolism, excretion and toxicity properties, including Lipinski rules were well measured by the following paraments: Pgp-inhibitor, Human Intestinal Absorption (HIA), 30 % Bioavailability (F_30 %), Plasma Protein Binding (PPB), Blood-Brain Barrier (BBB) Penetration, CYP1A2 inhibitor, CYP1A2 substrate, Clearance (CL), half-life (T_1/2), hERG Blockers and rat oral acute toxicity, and so on. Finally, this web tool was used to evaluate the drug-likelihood of aurone derivative 1c, including the Lipinski rule and bioavailability.

2.3.8. Statistical analysis

Dates were presented as means ± SD and analyzed by one-way-ANOVA by SPSS version 24.0. Results are considered as statistical significance when p < 0.05.

3. Results and discussion

3.1. Chemistry

In this paper, the benzofuranones intermediate approach was selected to synthesize polymethoxy aurones, which were easily prepared from commercially available 3, 4, 5-trimethoxy benzaldehyde (see Scheme 1).

Scheme 1.

Synthesis of polymethoxy aurones.

In this process, Dakin oxidation was first applied to synthesize compound 4 from 3, 4, 5-trimethoxybenzaldehyde. After the reaction, NaHCO₃ was added to neutralize the by-product 3-chlorobenzoic acid, which gives a good yield. Compound 4 was reacted with potassium hydroxide in methanol to produce 3, 4, 5-trimethoxyphenol within 5 min, otherwise the by-products appear and increase in quantity over time. Then, potassium carbonate/acetone system was applied to replace KOH or Na in the substitution reaction between 3, 4, 5-trimethoxyphenol and 2-chloroethylene acetate with good yields. Esterolysis reactions take place in the NaOH/EtOH system to obtain compound 3 without purification. Compound 2 was cycling from compound 3 through polyphosphoric acid (PPA) catalyze in anhydrous condition. The single geometric isomer (Z) compound 1 was prepared from compound 2 by KOH catalyzed condensation with an appropriate aldehyde, because of an intermolecular hydrogen bond between carbonyl and methine, which is generally more thermodynamically stable than the (E)-isomer [39].

3.2. Biological evaluation

3.2.1. In vitro anti-proliferative activity

All of the synthesized compounds were screened by the CCK-8 assay. Prostate cancer (DU145), breast cancer (MCF-7) and lung cancer (H1299) cell lines were selected to screen for anti-proliferative activities, and the results showed in Table 1 and Fig. 2. Compared with the MCF-7 and H1229 cell lines, compound 1b and 1c still have certain inhibition ability below the concentration of 50 μM and showed a certain degree of selectivity in DU145 cells. Then, the structure-activity relationship of aurone analogues in prostate cancer was discussed.

Table 1.

The IC₅₀ values of synthesized compounds on three cancer cells.

	IC50 (μM)
	DU145	MCF-7	H1299
1a	–	218.67	517.51
1b	37.68	57.04	＞1000
1c	15.56	104.38	＞1000
1d	933.89	941.51	＞1000
1e	>1000	814.07	＞1000

Fig. 2.

Invitro cytotoxicity analysis of the synthesized compounds against three different human cancer cell lines.

The results of the CCK-8 assay revealed that the introduction of halogen into the C ring enhance the anti-prostate cancer activity. Compound 1a has only one hydroxyl group on the C ring, which may be responsible for the lack of cytotoxicity in the DU145 cell line. In addition, the hydroxyl group may be synergistic with the halogen group of aurones, as compound 1b and 1c with both hydroxyl and halogen groups achieve better anti-prostate cancer activity than compound 1d and 1e with only the halogen group in the C ring.

Based on the above results, compound 1c showed the best anti-proliferative activity with IC₅₀ = 15.56 μM. Compound 1c was selected for further biological evaluation to reveal its anti-prostate cancer mechanism.

3.2.2. Network pharmacology study

Network pharmacology studies have been applied to predict the anti-prostate cancer targets of polymethoxy aurones. Compound 1c, the most cytotoxicity compound, was used for prediction. As Fig. 3A showed, a total of 20 potential targets of compound 1c were predicted by the ChemMapper database with the property of “Homo sapiens” set. Then, the GeneCards database was used for searching prostate cancer-related genes and ultimately 2130 genes (Fig. 3B) were retrieved according to the screening criteria of containing the keyword “prostate cancer” and a relevance score greater than or equal to 5. As shown in the Venn diagram of Fig. 3C, the 14 overlapped targets (both 1c and prostate cancer-related) were identified as key targets for the anti-cancer activity of 1c for prostate cancer. To further shed light on the biological mechanism of 1c against prostate cancer, KEGG pathway enrichment analysis was performed based on the 14 common targets with a cut-off P-value of 0.05 and count-value of 3.

Fig. 3.

The analysis of network pharmacology. (A) Compound-target network. The purple node represents compound 1c, the laurel green nodes are targets. (B) Disease-target network. The deep purple rectangle represents prostate cancer, and the blue-black parts are targets. (C) The Venn diagram of the common genes between prostate cancer targets (blue circle) and compound 1c (laurel green circle). (D) The top 5 pathways and their –Log10 (P-Value).

Finally, the top 5 potential pathways were selected and shown in Fig. 3D, which mainly include the thyroid hormone signaling pathway, measles, herpes simplex infection, prolactin signaling pathway, and cell cycle. Among the pathways, the cell cycle pathway appears to be closely related to the occurrence and development of tumors and has been identified as a prominent pathway for 1c against DU145 cells. Based on the above results, we design the cell cycle experiments to verify our hypothesis.

3.2.3. Effect on cell cycle progression

To investigate the impact of compound 1c on the cell cycle progression, DU145 cells were treated with compound 1c (5 and 10 μM) for 72 h and cell cycle distribution was assessed by 7-AAD-based flow cytometric analysis. As the results shown in Fig. 4A and B, significant changes in cell cycle characteristics of DU145 cells were observed after compound 1c treatment. In comparison with untreated control cells, the percent of cells at G0/G1 phase showed slightly decrease while the percentage of cells at G2/M treated with 5 μM or 10 μM of 1c were 31.36 % and 39.17 %, respectively, which were markedly increased. From the above studies, we can infer that compound 1c arrested the cell cycle at G2/M phase leading to anti-proliferative activity against prostate cancer.

Fig. 4.

Compound 1c induced G2/M−phase arrest in DU145 cells. (A) The distribution of cell cycle was monitored using flow cytometry after DU145 cells were treated with compound 1c for 72h. (B) Bar chart depicting the cell cycle distribution in DU145 cells. (C) The expressions of CyclinB1, CDK1, and p21 in DU145 cells were detected by Western blot. GAPDH was used as control. (D) Bar chart showing the relative expression of CyclinB1, CDK1, and p21 in DU145 cells. Data are expressed as mean ± SD, compared with the control group: *P < 0.05.

Growing evidence has demonstrated that cyclin-dependent kinases (CDKs) participated the regulation of DNA synthesis and phosphorylation process, and the CyclinB1/CDK1 complex was verified to modulate the transition of the cell cycle from G2 to M phase. [40, 41, 42] Hence, we further assessed the molecular mechanisms underlying the cell cycle arrest induced by compound 1c in DU145 cells. Several key proteins involved in the progression of G2/M in DU145 cells were then examined by western blotting to confirm that compound 1c was able to block the G2/M cell cycle to inhibit the proliferation of prostate cancer cell lines. p21 (also known as p21W AF1/Cip1) expression was detected additionally, because this gene is an important inhibitor of CDKs, which could inactivate the CyclinB1/CDK1 complex and then block the cell cycle in the G2/M phase. [43, 44] The results shown in Fig. 4C conform with our expectation that compound 1c can decrease the expression of CyclinB1 while up-regulated the expression of p21 in a dose-dependent manner, ultimately inducing G2/M-phase arrest.

3.3. Molecular docking

Cyclin B is the only CDK partner of CDK1, both of them can together control the extent of inhibitory phosphorylation within the CDK1 active site with phosphatases [45]. Alternatively, CKS proteins may enhance selective phosphorylation of CDK1 substrates at mitosis, suggesting that the use of the ternary Cyclin B1/CDK1/CKS complex may shed light on synergistic effects in the cell cycle [45]. Therefore, in this paper we employed the ternary complex of human protein (PDB code: 5HQ0) to operate the molecular docking to study the blocking cell cycle mechanism of compound 1c, expecting to exploit novel CDKs inhibitors in future.

In order to ensure the consistency between the docking model and the co-crystal protein, we first docked the native ligand (ID: LZ9), and the root mean square deviation (RMSD) value between the docked pose and the crystal conformation of the native ligand was found as 0.13 Å, which indicated the docking model was reasonably reliable [46]. Based on this reliable docking model, the docking compound 1c has a total score of 5.32, which is higher than other aurone derivatives (Table 2).

Table 2.

Docking results of aurone derivatives (PDB:5HQ0).

Compounds	1a	1b	1c	1d	1e
Total Score	4.65	4.53	5.32	5.21	5.27

The CDK1 protein divides into six regions, there are phosphate pocket, ribose pocket, solvent exposed area, DFG area, gatekeeper and hinge area. Among them, hinge area is closely associated with activity [47, 48]. The activation segment in the hinge region can form a platform that recognizes CDK substrate residues to either side of the phosphotransfer site, which is the key factor inhibiting CDK protein [33]. Therefore, lots of CDK inhibitors form hydrogen bonds with this activation segment [49]. When docking with compound 1c, the result (Fig. 5) shows that compound 1c in the ATP binding pocket forms one hydrogen bond with the amino acid residue LEU83 in the activation segment of CyclinB1/CDK1/CKS protein via the carbonyl group in the core furanone fragment [50]. However, this binding is weaker compared to the native ligand. The native ligand forms not only two hydrogen bonds with LEU83, but also one hydrogen bond with GLU81, which is also in the active segment [51]. Maybe that is the reason why the native ligand obtains a better total score (total score = 7.90) than compound 1c. In the similar way, except for compound 1c, other aurone derivatives do not form hydrogen bonds with LEU83 (see supporting materials). It can be concluded that different substitutions on the C ring affect the conformation of the active pocket, and only the appropriate conformation will interact with the active segment to produce biological activity.

Fig. 5.

Molecular docking Interaction of compound 1c with Cyclin B1/CDK1/CKS complex proteins, hydrogen bonds were shown as yellow dotted lines.

Combined with the CCK8, cell cycle assay and molecular docking results, we can infer that compound 1c is a potential CyclinB1/CDK1 inhibitor that can bind to hinge region of the protein and block the G2/M cell cycle, thereby inhibiting the proliferation of prostate cancer cells.

3.4. Cellular thermal shift assay

Through molecular docking, we have verified that compound 1c can bind to the Cyclin B1/CDK1/CKS complex protein theoretically, now, CETSA, which is a valuable tool for the validation of drug-target engagement, has applied to confirm the results by practice [52]. Target proteins usually become more stable when they bind to drug molecules. Under general conditions, the protein will be degraded with the increase of temperature, but when the protein binds to the drug, the amount of under-graded protein will increase at the same temperature, so the hot melt curve of the composite protein will shift to the right compared with the solvent control group [53].

Compared with the control group, the degraded temperature of CyclinB1 and CDK1 that binding compound 1c was raised obviously (Fig. 6). In control group, when the temperature rises to 62 °C, the CyclinB1 degraded observably, and the CyclinB1 composite protein that binding compound 1c just degraded at the temperature 67 °C. There was the same phenomenon as the CDK1, the degrade temperature of pure CDK1 was approximately 5° lower than its composite protein, whereas the reference protein GAPDH had the same degrade temperature whether composite protein or not. Therefore, these results verified that the compound 1c has already bonded to the CyclinB1/CDK1, and the compound 1c is a potential CyclinB1/CDK1 inhibitor, however the binding does not tightly enough. This result is consistent with the moderate total score of compound 1c in molecular docking.

Fig. 6.

The 1c-CyclinB1/CDK1 binding was examined in DU145 cells using cellular thermal shift assay and Western blot. The DU145 cells proteins were incubated with (A) DMSO and (B) 1c (200 μM) at room temperature for 1 h.

3.5. ADMET prediction

ADMET properties was very important index that evaluated the drug-like properties and pharmacokinetics of drug candidates. We used ADMET lab 2.0 to evaluate the ADMET properties of compound 1c [54]. The results show that compound 1c satisfies the Lipinski rule with a molecular weight of 406.01, LogP of 3.237, number of hydrogen bond donors of 1 and number of hydrogen bond acceptors of 6. The absorption is good, except for the Pgp-inhibitor parameter, which is poor, but the other absorption parameters are up to standard. In the distribution, the volume distribution is 0.482, which is in the optimal range where compound 1c cannot cross the blood-brain barrier. In metabolism, compound 1c is a CYP1A2 inhibitor to a large extent, reaching a value of 0.942. The closer the value is to one, the more likely it is to be an inhibitor. However, compound 1c is appropriately a short half-life compound with T_1/2 of only 0.358 and poor clearance. Compound 1c had a lower risk of cardiac toxicity, the value is 0.006, and moderate oral acute toxicity in rats with a value of 0.673. A value closer to 0 is more likely to have no toxicity.

The detail of ADMET results please see the supporting information. The ADMET predictions point to future directions of structural modification, with the distribution and excretion terms being the main ones.

4. Conclusion

A convenient method was used to synthesize five 4,5,6-trimethoxy aurone derivatives, and activity screening showed that these polymethoxy aurones had particular inhibitory effect on prostate cancer cells. Structure-activity relationship studies have suggested that the cytotoxicity of aurone analogues on DU145 cells may be related to a synergistic interaction between the halogen and hydroxyl groups on the C ring. Network pharmacological predictions suggest that polymethoxy aurones 1c inhibits prostate cancer cells through cell cycles. To verify this finding, we performed cell cycle and Western blot experiments. The results pointed out that the compound 1c induced G2/M-phase arrest in DU145 cells by regulating the expression of CyclinB1 and p21. Next, we investigate the mechanism of action between CyclinB1/CDK1 and compound 1c. Combining with molecular docking and CETSA techniques, compound 1c was found to be able to occupy the ATP binding cavity of CyclinB1/CDK1/CKS, interacting with the key amino residues LEU83 in the activation-segment responsible for influencing the cell cycle of prostate cancer. At last, applied ADMET prediction to evaluate the draggability of compound 1c. In brief, compound 1c is a potential CyclinB1/CDK1 inhibitor, and in the future, we can enhance its binding to the active segment of CyclinB1/CDK1/CKS protein, improve its distribution and excretion properties, to develop novel aurone derivative CDK1 inhibitors.

Funding statement

This work was supported by the Guangxi Science and technology project (FANGKE ZY20221502), Guangxi Natural Science Foundation (2020GXNSFAA297114, 2015GXNSFBA139028), and Advanced Innovation Teams and Xinghu Scholars Program of Guangxi Medical University.

Data available

Data included in article/supp. material/referenced in article.

CRediT authorship contribution statement

Zheng Wu: Data curation, Formal analysis, Funding acquisition, Project administration, Software, Writing – original draft, Writing – review & editing. Yaoyao Han: Data curation, Investigation, Software, Writing – original draft. Xiaolan Li: Data curation, Software, Writing – review & editing. Qiuping Zhang: Formal analysis. Renjin Deng: Data curation. Hong Ren: Data curation. Wenjing He: Data curation. Xinduo Wu: Data curation, Methodology. Hongwei Guo: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing – review & editing. Dan Zhu: Funding acquisition, Project administration, Supervision, Writing – review & editing.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Hongwei Guo reports financial support was provided by the Advanced Innovation Teams and Xinghu Scholars Program of Guangxi Medical University. Dan Zhu reports financial support was provided by Guangxi Natural Science Foundation (2020GXNSFAA297114). Zheng Wu reports financial support was provided by Guangxi Natural Science Foundation (2015GXNSFBA139028).

Appendix A. Supplementary data

The following are the Supplementary data to this article: